EP3126514A1 - A combination product comprising a nucleic acid structure and a single-stranded nucleic acid molecule - Google Patents
A combination product comprising a nucleic acid structure and a single-stranded nucleic acid moleculeInfo
- Publication number
- EP3126514A1 EP3126514A1 EP15715207.5A EP15715207A EP3126514A1 EP 3126514 A1 EP3126514 A1 EP 3126514A1 EP 15715207 A EP15715207 A EP 15715207A EP 3126514 A1 EP3126514 A1 EP 3126514A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- nucleic acid
- combination product
- stranded
- ligand
- acid molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
Definitions
- a combination product comprising a nucleic acid structure and a single-stranded nucleic acid molecule
- the present invention relates to a combination product which can be used to detect and/or characterize interactions between two molecules, in particular two proteins and most particular between an antigen and an antibody.
- the combination product comprises a nucleic acid structure comprising a double-stranded part and a single- stranded part which is capable of hybridizing to another single-stranded nucleic acid molecule which is also part of the combination product.
- SPR surface plasmon resonance
- switchSENSE Another recently developed technique is the "switchSENSE” technology which is based on the electrical actuation of nucleic acid double strands to which an interactant is attached at high frequency on microelectrodes. The orientation of the nucleic acid double strands is then monitored by time -resolved single photon counting. If an analyte binds to the interactant attached to the nucleic acid double strand, the switching dynamics of the double strand is slowed down in a
- the interactant which is immobilized on the biosensor surface is typically randomly distributed, leading to differences in the local density of the interactant on the surface. Due to these differences the spatial arrangement on the surface of interactants such as antigens which bind to an antibody to be analyzed cannot be controlled. Consequently, when bivalent or bispecific antibodies are analyzed, a certain percentage of the antibodies will bind the antigen via only one Fab arm, if at the binding site no second antigen is available for binding, while another percentage of the antibodies will bind the antigen via both Fab arms, if two antigens are located close to each other on the biosensor surface so that the antibody can bind to both of them. This heterogenous mixture of antibodies bound via one Fab arm and via two Fab arms leads to complex signals which are difficult to analyze.
- the present invention provides a combination product which enables the exact spacing between two molecules and thereby an accurate analysis of the interaction between ligands and their binding partner, in particular the interaction of antigens with multivalent or multispecific antibodies such as bivalent or bispecific antibodies.
- the present invention provides a combination product comprising:
- nucleic acid structure having a sequence selected such that it forms at least one double strand (4) and at least one single-stranded loop (1);
- the nucleic acid structure is a stem-loop structure.
- nucleic acid structure formed by three separate single-stranded nucleic acid molecules, wherein the first (13) and the second (14) single-stranded nucleic acid molecule each form a double strand with a first and a second part of the third single- stranded nucleic acid molecule (15) and wherein a third part of the third single- stranded nucleic acid molecule which is located between the first and the second part forms a single-stranded loop; and - -
- the combination products of the present invention may comprise one single-stranded nucleic acid molecule (6) which is capable of hybridizing to and forming a double strand with the single-stranded loop and to which a first ligand (7) is attached to the 5' end of (6) and a second ligand (8) is attached to the 3' end of (6).
- the combination products of the present invention may comprise a first (11) and a second (12) single-stranded nucleic acid molecule, wherein the first and the second nucleic acid molecules (11,12) are capable of hybridizing to and forming a double strand with different parts of the single-stranded loop (1) and wherein a first ligand (7) is attached to the first nucleic acid molecule (11) and a second ligand (8) is attached to the second nucleic acid molecule (12).
- the first ligand (7) and the second ligand (8) may be the same or different.
- the at least one ligand (7,8) is a protein or part thereof.
- the nucleic acid structure is attached to a substrate (10).
- nucleic acid structure is labeled with a fluorophor (2).
- the present invention also provides a substrate to which the combination product of the present invention is attached and a kit comprising the combination product or the substrate and instructions for its use.
- the combination product or the substrate of the present invention may be used for the detection and/or characterization of the interaction of a ligand with at least one binding partner thereof, for protein purification or for the diagnosis of diseases.
- the present invention provides a method for detecting and/or characterizing molecular interactions comprising:
- the present invention provides a method for detecting and/or characterizing molecular interactions comprising:
- the ligand is an antigen and the binding partner is an antibody, more preferably the antibody is a bispecific antibody.
- the combination product or the nucleic acid structure is attached to a substrate and is labeled with a fluorophor and the interaction is detected and/or characterized by the steps of:
- the combination product is attached to a substrate, the substrate has an electrode, the combination product is labeled with a fluorescent dye and the interaction is detected and/or characterized by the steps of:
- the oscillating molecules shall be understood as the combination product which comprises the nucleic acid structure and the at least one single-stranded nucleic acid molecule which has hybridized to the nucleic acid structure.
- the oscillating molecules are the molecules of the moved combination product.
- Said combination product can be switched by means of the external electrical field from a lying state into a standing state and vice-versa. This measurement principle can be gathered from, for example, the scientific publication "Detection and Size Analysis of Proteins with Switchable DNA Layers", Nano Letters, 2009, Vol. 9, 1290 - 1295.
- a dynamical method for detecting binding interactions By causing the movement of the combination product from the lying into the standing position, a dynamical method for detecting binding interactions is provided. This method facilitates the detection based on a change of the dynamical behaviour of the combination product due to the binding of a binding partner to a ligand of the combination product.
- These aspects of the present invention make use of the fact that the combination product is electrically charged and thus has a polarity. More details hereof will be explained in the context of the Examples and Figures hereinafter. In other words, in this embodiment the herein presented method makes use of the _ _
- Switchsense technology as has been described before. In further embodiments of this method the detection is based on an observed change of a hydrodynamic resistance of the switched molecules. Further details thereof will be explained in the context of the Figures, particularly in the context of Figures 4 and 5.
- quenching the signal of the label is used, wherein the quenching mechanism used depends on the distance between the quenching medium and the label.
- the used quenching is distant dependent.
- a quenching layer which is used also as an electrode, may be provided to facilitate the quenching of electromagnetic radiation emission from the label.
- An applied bias polarizes the electrode, leading to the formation of a Gouy- Chapman-Stern screening layer.
- Non-radiative energy transfer from the label to surface plasmons in the quenching layer may quench the emitted signal intensity when the label approaches the surface. Therefore, high signal intensities indicate a standing combination product configuration and less quenching during negative applied electrode bias. Low signal intensities indicate a lying combination product configuration during positive applied electrode bias. The latter is the case for a quenching layer being close to the substrate.
- said quenching layer may also be positioned opposite of the electrode, e.g. on the counter electrode.
- the electrode is a metal, a photoluminescent (PL) label which emits low
- the label may be selected according to the following aspects.
- the absorption spectrum of the quenching medium coincides to some extent with the emission spectrum of the label, such that non-radiative energy transfer is possible.
- the label may be a PL emitter and can be an organic dye molecule or also a nanoparticle.
- the present invention also related to a method for separating a target protein from impurities comprising the steps of:
- Figure 1 Schematic drawing of a first representative combination product of the present invention
- the nucleic acid structure also called slingshot structure
- One of the nucleic acid strands forming the double strand (4) is attached to a substrate (10) by a linker (5).
- a double strand (9, also called spacer bar) forms between the single-stranded loop and the single-stranded nucleic acid molecule.
- the nucleic acid structure is the same as in a).
- Each of the two separate single- stranded nucleic acid molecules (11, 12) to each of which a ligand (7, 8) is attached forms a double strand with the single-stranded loop.
- FIG. 2 Schematic drawing of a second representative combination product of the present invention a) Two single-stranded nucleic acid molecules (13, 14, also called handle 1 and handle 2) are each attached to a substrate (10) by a linker (5). A third single-stranded - - nucleic acid molecule (15; also called connector) forms a double strand with both single-stranded nucleic acid molecules (13, 14) and a single-stranded loop (16), thereby creating a structure called "triangular arch". Each of the two separate single- stranded nucleic acid molecules (1 1 , 12; also called c-spacers) to each of which a ligand (7, 8) is attached forms a double strand with the single-stranded loop.
- a small single-stranded nucleic acid molecule without a ligand (17) is hybridized to a part of the single-stranded loop in addition to the c-spacers.
- Figure 3 Formation of double strands by hybridization of a single-stranded nucleic acid molecule to the single-stranded loop of the nucleic acid structure a) Hybridization of complementary spacer strand (c-spacer) to the sling in solution monitored by FRET.
- the central nucleotides of sling and c-spacer were labelled with Cy3 (green, donor) and Cy5 (red, acceptor) dyes, respectively.
- Cy3 green, donor
- Cy5 red, acceptor
- the solid line is - - a single exponential fit, from which the rate constant konobs of hybridization is analyzed.
- kon obs as a function of c-spacer concentration in solution.
- Regeneration repeated removal of c-spacer sequence from immobilized slingshot structures by pH13 denaturation and re-hybridization with fresh c-spacer.
- Figure 4 Binding of full-length antibodies and antibody fragments to the combination product wherein either two identical or two different antigens are attached to the c-spacer molecule.
- the Dynamic Response parameter (DR, area under the time -resolved fluorescence trace) is a number representing the switching velocity; high DR values denotes fast switching.
- DR Dynamic Response parameter
- DR values are normalized by the DR of combination products not bound by antibody.
- Figure 5 Analysis of dissociation kinetics a) Dissociation of anti-biotin IgG from a monovalent antigen probe or two monovalent antigen probes.
- the thin continuous line is a double-exponential fit, thick gray lines are plots of the two individual exponential components.
- the continuous line is a single exponential fit.
- Figure 6 Analysis of possible binding conformations by varying the length of the single-stranded nucleic acid molecule. a) At a length of about 20 nucleotides (corresponding to 6 nm) the bivalent binding of the antibody to both ligands of the c-spacer is possible. b) At a length of about 30 nucleotides (corresponding to 9 nm) the bivalent binding of the antibody to both ligands of the combination product is still possible, but stressed due to a stretched conformation and a lower binding energy. c) At a length of about 40 nucleotides (corresponding to 12 nm) the bivalent binding of the antibody to both ligands of the combination product is not possible.
- Figure 7 Formation of the "triangular arch" structure (see Figure 2) and hybridization of the structure with a single-stranded c-spacer a) Hybridization of the connector strand to the surface-immobilized handles 1 and 2 followed in real-time by fluorescence measurements. The open symbols show the fluorescence intensity at +0.4 V and the filled symbols show the fluorescence intensity at -0.4 V. b) Hybridization of the single-stranded c-spacer to the single-stranded loop formed by the connector strand followed in real-time by fluorescence measurements. The open symbols show the fluorescence intensity at +0.4 V and the filled symbols show the fluorescence intensity at -0.4 V.
- Figure 8 Binding of a full-length anti-biotin antibody to the "trinagular arch” structure (see Figure 2).
- the Dynamic Response parameter (DR, area under the time- - - resolved fluorescence trace) is a number representing the switching velocity; high DR values denotes fast switching.
- combination product is intended to mean that the product contains at least two components, i.e. a nucleic acid structure and a single-stranded nucleic acid molecule which in the combination product is either hybridized to the nucleic acid structure by forming a double strand with the single-stranded loop or remains separate.
- nucleic acid structure is a secondary structure formed by one or more nucleic acid molecules.
- a first embodiment also called “slingshot” which is illustrated in Figure 1 it is formed by one nucleic acid molecule having a sequence selected such that it forms at least one double strand and at least one single-stranded loop, for example a stem-loop structure.
- nucleic acid structure is formed by three separate single-stranded nucleic acid molecules, wherein the first and the second single-stranded nucleic acid molecule each forms a double strand with a first and a second part of the third single-stranded nucleic acid molecule and wherein a third part of the third single-stranded nucleic acid molecule which is located between the first and the second part forms a single- stranded loop.
- the following discussion applies to both the first and the second embodiment of the nucleic acid structure.
- nucleic acid refers to any type of nucleic acid molecule such as DNA or R A or PNA or LNA with DNA being the preferred nucleic acid.
- hybridizes to is intended to mean that the single-stranded nucleic acid molecule forms a double strand with the single-stranded loop of the nucleic acid structure by the interaction of
- complementary bases in the single strands The person skilled in the art knows how to select a sequence such that it forms a double strand and a single-stranded loop.
- a nucleotide double-strand forms by forming hydrogen bonds between complementary bases, i.e. between adenine and thymine or between cytosine and guanine.
- the molecule which forms a double strand has to contain a first sequence and a second sequence complementary to the first sequence.
- the first single-stranded nucleic acid molecule has to be complementary to the first part of the third single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule has to be complementary to the second part of the third single- stranded nucleic acid molecule.
- the double strand forms if the two sequences are contacted under suitable conditions, i.e. a pH above 2, a salt concentration equivalent to more than 20mM NaCl and a temperature below the melting temperature of the double strand which is dependent on the length of the double strand.
- Suitable conditions for forming a double strand are also known to the skilled person and include the use of a buffer comprising 40 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4 and 0.05% Tween 20 at a temperature of 25°C.
- sequences forming the double strand do not have to be 100% complementary to each other, meaning that in the double strand one or more bases may not be paired with their complementary base.
- the person skilled in the art knows that longer double-stranded molecules permit a larger number of non-pairing bases than shorter double-stranded molecules without significantly influencing the stability of the double strand.
- at least 50%, preferably at least 60% and more preferably - - at least 70% of the bases within the sequences forming the double strand should be complementary to each other.
- nucleic acid structure For the sequence to form a single-stranded loop it is necessary that the sequence within the loop does not show significant complementarity to the other sequences within the nucleic acid structure so that no double-stranded structures are formed between the sequence within the loop and the first or second sequence of the double strand.
- additional nucleotides may be present between the sequences forming the double strand and the sequence forming the single-stranded loop which nucleotides are not part of the double strand of the nucleic acid structure and which also do not take part in the formation of the double strand between the single-stranded loop and the single-stranded nucleic acid molecule.
- the sequence connecting the double strand(s) with the single-stranded loop is a sequence consisting of only one kind of nucleotide, preferably the sequence consists exclusively of thymidine nucleotides.
- the insertion of this connecting sequence may help to avoid base-stacking interactions and to allow for flexibility.
- the length of the sequence connecting the double strand(s) with the single-stranded loop depends on the length of the double strand formed by the single-stranded loop and the single-stranded nucleic acid molecule and may be up to 50%> of the length of this double strand.
- the nucleic acid structure is to be attached to a substrate, one or both of the sequences forming the double strand may be attached to a linker which facilitates the attachment to the surface.
- Suitable linker molecules are known to the skilled person and depend on the surface to which the nucleic acid structure is to be attached.
- the linker is preferably a thiol linker such as (CH 2 )6-SH or di-thiol phosphoramidite.
- a silane linker may be used.
- the linker may be biotin.
- Nucleic acid molecules with an attached linker can be obtained commercially from companies such as Euro fins Genomics or atdbio.
- the nucleic acid structure is a stem-loop structure that has one single-stranded loop which is associated with one double-stranded stem.
- stem-loop structure that has one single-stranded loop which is associated with one double-stranded stem.
- the double strand within the nucleic acid structure may have a length of 5 to 300, preferably 10 to 200 or 15 to 150, more preferably 18 to 120 or 20 to 100, even more preferably 23 to 80 or 25 to 60 and most preferably 30 to 50 base pairs.
- the double strand may consist of DNA, RNA, PNA and LNA homoduplexes and heteroduplexes thereof such as DNA/RNA, DNA/PNA, DNA/LNA, RNA/PNA and PNA/LNA.
- the skilled person knows that the kind of nucleic acid used may influence the stability of the double strand so that the minimal length required for the formation of a stable double strand depends on the type of nucleic acid forming the double strand.
- the single-stranded loop may have a length of 10 to 170, preferably of 11 to 150 or 12 to 120, more preferably of 13 to 120 or 14 to 100, even more preferably of 15 to 90 or 16 to 70, particularly preferably of 17 to 50 or 18 to 40 and most preferably of 20 to 30 nucleotides. Similar to the double strand the single-stranded loop may consist of DNA, RNA, PNA and LNA.
- the at least one single-stranded nucleic acid molecule which is capable of forming a double strand with the single-stranded loop may have a length of 10 to 170, preferably of 11 to 150 or 12 to 120, more preferably of 13 to 120 or 14 to 100, even more preferably of 15 to 90 or 16 to 70, particularly preferably of 17 to 50 or 18 to 40 and most preferably of 20 to 30 nucleotides.
- the single-stranded nucleic acid molecule may consist of DNA, RNA, PNA and LNA. It is not required that the single-stranded nucleic acid consists of the same type of nucleic acid as the double strand or the single-stranded loop. - -
- the sequence of the single- stranded nucleic acid molecule(s) has to be complementary to the sequence of the single-stranded loop.
- the sequences forming the double strand do not have to be 100% complementary to each other, meaning that in the double strand one or more bases may not be paired with their complementary base.
- longer double-stranded molecules permit a larger number of non-pairing bases than shorter double-stranded molecules without significantly influencing the stability of the double strand.
- at least 50%, preferably at least 60% and more preferably at least 70% of the bases within the sequences forming the double strand should be complementary to each other.
- the single-stranded nucleic acid molecule may have the same length as the single-stranded loop, as long as the single-stranded nucleic acid molecule is able to form a stable double strand with the single-stranded loop by hybridizing to each other.
- the single-stranded loop may comprise nucleotides which do not form part of the double strand with the single-stranded nucleic acid molecule.
- the single-stranded nucleic acid molecule may comprise nucleotides which do not form part of the double strand with the single-stranded nucleic acid loop.
- the single-stranded nucleic acid molecule does not comprise nucleotides capable of forming a double strand with the double strand (4) of the nucleic acid structure of the first embodiment or with the double strand formed between the first (13) and the second (14) single-stranded nucleic acid molecule and the first and second part of the third nucleic acid molecule in the nucleic acid structure of the second embodiment.
- the double strand (4) within the nucleic acid structure of the first embodiment is not disrupted, i.e. it remains intact.
- the double strand formed between the first (13) and the second (14) single-stranded nucleic acid molecule and the first and second part of the third nucleic acid molecule is not disrupted, i.e. it remains intact, upon formation of the double strand between the single-stranded nucleic acid molecule(s) and the single-stranded loop.
- the combination product comprises one, i.e. a single, single- stranded nucleic acid molecule which is capable of hybridizing to and forming a double strand with the single-stranded loop.
- a ligand is attached to each end of the single-stranded nucleic acid molecule, i.e. the 5' and the 3' end.
- the length of the single-stranded nucleic acid molecule determines the distance between the two ligands attached to it. Hence, it is possible to vary the distance between the two ligands by varying the length of the single-stranded nucleic acid which enables the analysis of spacing between binding sites in the molecule interacting with the ligand and of the influence of the distance on the binding energy.
- the combination product comprises two separate single-stranded nucleic acid molecules, each of which is attached to one ligand.
- the two single-stranded nucleic acid molecules are capable of hybridizing to separate, non-overlapping parts of the single-stranded loop within the nucleic acid structure. It is not necessary that the two nucleic acid molecules together bind over the whole length of the single-stranded loop. It is also not necessary that the two nucleic acid molecules bind adjacent to each other, meaning that a part of the single-stranded loop may not be bound by any of the single-stranded nucleic acid molecules to form a double strand, but remains single-stranded.
- the length of the single-stranded loop sequence to which both single-stranded nucleic acid molecules hybridize determines the distance between the ligands attached to the separate nucleic acid molecules. If the two single-stranded nucleic acid molecules hybridize to adjacent parts of the single-stranded loop, the distance between the two ligands attached to the single-stranded nucleic acid molecules is the sum of the length of these two single-stranded nucleic acid molecules.
- a third single-stranded molecule may be hybridized to those nucleotides in the single-stranded loop which are located between _ _ the nucleotides to which the two single-stranded nucleic acid molecules hybridize.
- This third single-stranded nucleic acid molecule allows to modulate the distance between the ligands attached to the two single-stranded nucleic acid molecules without having to modulate these molecules themselves.
- ligands such as proteins to nucleic acid molecules.
- One possibility is to modify the nucleic acid molecule by 6-maleimidohexanoic acid N-hydroxysuccinimide ester and then to react the modified DNA with the protein having suitable amino acid residues for conjugation (see, e.g., Fujiwara et al. (1988). J. Immunol. Methods 112: 77-83;
- kits are available for producing protein-nucleic acid conjugates, for example from Solulink. An overview of different conjugation techniques is provided in Hermanson,
- a “ligand” is a molecule which can bind to another molecule by non-covalent bonds due to a specific affinity to the other molecule.
- the term "ligand” is intended to include, but not being limited to, proteins or parts thereof as well as non-proteinaceous chemical molecules which have to have a size which does not interfere with the formation of the double strand.
- the ligand is a protein or part thereof.
- the part of the protein may have a minimum length of at least six amino acids which is the minimum length of an epitope which is recognized by an antibody.
- the first and the second ligand attached to the single-stranded nucleic acid molecule may be the same, i.e. they may have the same chemical structure. For example, if the interaction of a bivalent, monospecific full-length antibody with an antigen is to be analyzed, the first and the second ligand may be this antigen.
- the first and the second ligand attached to the single- stranded nucleic acid molecule may be different, i.e. they may have a different chemical structure.
- different proteins or parts of different _ . proteins may be used as first and second ligand.
- the first ligand may be the first antigen or part thereof and the second ligand may be the second antigen or part thereof, wherein the first and the second antigens are the target antigens of the bispecific antibody.
- different parts of the same protein may be used as first and second ligand which for example allows determining the binding epitope of an antibody.
- first and the second ligand may have the same amino acid sequence, but may differ in post-translational modifications such as phosphorylations, methylations and acetylations.
- This embodiment allows the analysis of the influence of post-translational modifications on the binding of a binding partner such as the binding of an antibody to its target antigen.
- the first ligand may also be a small molecule and the second ligand may be a protein or part thereof. Such an embodiment may be used to investigate the allosteric inhibition of an enzyme which is the binding partner by a small molecule. If the binding partner to be analyzed with the combination product of the present invention is a signal transduction molecule, the first ligand may be a protein which is supposed to be upstream of the binding partner in the signal transduction cascade and the second ligand may be a protein which is supposed to be downstream of the binding partner in the signal transduction cascade.
- the nucleic acid structure part of the present combination product may be attached to a substrate.
- substrate is to be understood in its broadest sense as a structure to which a nucleic acid molecule can be coupled reversibly or irreversibly and includes, for example, planar matrices such as chips or beads.
- the substrate may be made of any substance, like e.g. glass, to which a nucleic acid molecule can be attached and may be made, e.g. from gold or other metals.
- a biochip may be used which comprises a film on top of said substrate.
- a gold film of 5-300 nm thickness may be used on a glass substrate or an organic layer, e.g. a conducting polymer or a dye-sensitized matrix may be used on the substrate. This layer may function as a quenching layer used in an embodiment of the detection method.
- a linker molecule such as a thiol may be required which is attached to the nucleic acid structure and enables the binding of the nucleic acid structure to the substrate by a chemical reaction.
- the chemical linker is chosen from the group that contains one of -or a combination of- the following reactive groups: aldehyde, ketone, thiol, amine, carboxyl, hydrazine, hydrazide, hydroxyl, glycan, azide, alkyne, alkene, silicon, and any combination thereof.
- the substrate is a gold surface to which the nucleic acid structure of the combination product is coupled by means of a thiol linker which is attached to and or all of the nucleic acid molecules forming the double strand.
- the present invention also relates to a substrate to which at least one combination product of the present invention is attached via the nucleic acid structure.
- To the substrate two, three, four, five, six, seven, eight, nine or ten, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 combination products may be attached.
- the nucleic acid structure and the single-stranded nucleic acid molecule are the same in all combination products attached to the substrate and the
- combination products will only differ in the first and/or the second ligand attached to the single-stranded nucleic acid molecule. If two separate single-stranded nucleic acid molecules are used in the combination product, it is possible to use the same first ligand in all combination products attached to the substrate and to vary only the second ligand within the substrate. In this way, many different molecule interactions may be analyzed in parallel by varying only one component of the combination product.
- the substrate with the nucleic acid structure attached to it can be used for the analysis of further interactions after removing the single-stranded nucleic acid molecule from the nucleic acid structure.
- the single-stranded nucleic acid molecule - - can for example be removed by treatment with a strongly basic solution such as a solution with pH 13 which denatures the double strand between the single-stranded loop and the single-stranded nucleic acid molecule.
- the single-stranded loop After removing the first single- stranded nucleic acid molecule or set of single-stranded nucleic acid molecules from the nucleic acid structure, the single-stranded loop can be hybridized with single- stranded nucleic acid molecules having other ligands attached to them.
- the present inventors have shown that the nucleic acid structure can be re-used at least 16 times without significantly influencing the quality of the analysis. If the combination product of the present invention is to be used for example in the "Switchsense" method, the nucleic acid structure may be labeled with a fluorescent dye.
- Suitable fluorescent dyes are known to the skilled person and include FITC, fluoresceine, rhodamine green, rhodamine red, cyanine3 and cyanine5.
- cyanine 3 and/or cyanine5 are used.
- Nucleic acid molecules which are labeled with fluorescent dyes can be obtained from different suppliers such as Euro fins Genomics or Genscript.
- the fluorescent dye may be located both in the single-stranded loop and in the double strand.
- the fluorescent dye may be located in the first and/or second single-stranded nucleic acid molecule or in the single-stranded loop.
- the combination product of the present invention can be used to detect and/or analyze interactions between molecules, in particular the interaction of one or more antigens with an antibody.
- detecting means that it can be determined whether such an interaction indeed exists or not.
- the characterization of an existing interaction includes the determination of parameters such as affinity and/or avidity of the molecules to each other, the binding kinetics, i.e. the kinetics of association and dissociation between two molecules, the size of the molecules, conformational changes in the three-dimensional structure of the molecules, chemical modifications changes in the charge of the molecules, binding energies (free energy, enthalpy, entropy) in equilibrium, energy barriers involved in association or dissociation processes and the like.
- the combination product of the present invention can also be used in the purification of proteins, i.e. the separation of proteins from other proteins and impurities present in a sample.
- the nucleic acid structure of the combination product may be attached to beads such as polystyrene or magnetic beads and the single-stranded loop forms a double strand with the single-stranded nucleic acid molecule. After incubation of the attached combination product with a sample containing the protein to be purified, the beads may be washed under suitable conditions to remove impurities and then the purified protein may be released from the beads.
- the combination product of the present invention can be used in the diagnosis of diseases in which two or more proteins bind to each other in a defined distance to form agglomerates. In this case, the combination product is construed to bind only to the proteins forming the agglomerates, but not to protein monomers or multimers in which the proteins have another distance to each other.
- the present invention is described with particular reference to the analysis of antigen/antibody interactions by the so-called "switchSENSE” technology. However, it becomes apparent from the present description that the present invention can also be used to detect and/or analyze interactions of other molecules by other detection techniques such as surface plasmon resonance.
- the biochip consists of a glass substrate (27 x 40 mm) with eight holes (1 mm diameter) which serve as in- and outlets for four flow channels.
- Au work electrodes 120 ⁇ diameter
- Pt counter electrodes were arranged in 4 areas with 6 electrodes each and fabricated by standard optical lithography and metallization techniques.
- Thiolated 48mer oligonucleotides were end-grafted to the gold electrodes via spotting with a pico-liter dispensing system in immobilization buffer (10 mM Tris pH 7.4, 200 mM NaCl, 1 ⁇ DNA).
- immobilization buffer 10 mM Tris pH 7.4, 200 mM NaCl, 1 ⁇ DNA.
- the sequence of the Cy3 labelled ss-DNA for immobilization was 5' HS-(CH 2 ) 6 -TAG TCG TAA GCT GAT ATG GCT GAT TAG TCG GAA GCA TCG AAC GCT GAT-Cy3 3' (Metabion, Germany). After 10 min incubation, the chip was assembled by using double adhesive film with die-cut flow channels as an intermediate layer and a cover slide as a top layer.
- the flow channels were 60 ⁇ high and 1 mm wide and covered one of four electrode areas each.
- the DNA-modified Au electrodes were passivated and unspecifically bound DNA was removed by co-adsorbing mercaptohexanol (ImM in 'T'-buffer: 10 mM Tris pH 7.4, 50 mM NaCl) for 30 min. After installation in the setup, the DNA layers were hybridized with complementary DNA and the density was adjusted using a previously reported electrical desorption procedure 40 .
- AF/F the relative switching amplitude
- F fluorescence at repulsive potential
- AF/F the relative switching amplitude
- negative voltage pulses e.g. -0.8V vs. Pt
- DNA was electrically desorbed from the surface until AF/F became maximal, indicating free DNA movement in a very dilute layer (density estimation ⁇ 10 10 molecules/cm 2 ).
- double stranded DNA layers were denatured by flowing NaOH solution (pH 12-13) over the electrodes for 10s.
- the layers could then be regenerated by hybridization with fresh complementary DNA (50 nM in T-buffer).
- a flow rate of ⁇ /min was typically used during sensing experiments, which was high enough to operate the sensor in the reaction-limited kinetics regime. An effect of the applied hydrodynamic flow on the DNA switching behaviour was not observed. - -
- nucleic acid structure according to the first embodiment of the present invention is also called slingshot or slingshot structure.
- the double strand within this structure is also called handle and the single- stranded loop is also called sling.
- the single-stranded nucleic acid molecule is also called c-spacer and the double strand formed between the single-stranded loop and the single-stranded nucleic acid molecule is also called spacer bar.
- Slingshot layers were prepared in the same way as standard layers.
- the slingshot sequences were: Handle and sling: (90 mer) 5 * Thiol- TAG TCG CCC GCT GAT ATG GCT GAT TCG TCG TTT TTG CAG CTG GT*G TCG AGA CGG GTT TTT CGA CGA ATC AGC CAT ATC AGC GGG CGA CTA -3 * ; spacer bar: (20mer) 5 * -CCC GTC TCG ACA CCA GCT GC -3 * .
- antigen l biotin
- antigen2 digoxigenin
- antigen l biotin
- antigen2 biotin
- antigen l digoxigenin
- antigen2 digoxigenin
- Triangular arch layers For the purpose of the following discussion the nucleic acid structure according to the second embodiment of the present invention is also called triangular arch structure.
- the immobilized single-stranded nucleic acid molecules are also called handles and the single- stranded nucleic acid molecule hybridizing to the handles forming a double strand is also called connector strand.
- the single-stranded nucleic acid molecule is also called spacer or c-spacer and the double strand formed between the single-stranded loop and the single-stranded nucleic acid molecule is also called spacer bar.
- Triangular arch layers were prepared in the same way as standard layers.
- a DNA strand called immobilization connector is hybridized to the single-stranded handle 1 and handle 2 before immobilization.
- the immobilization connector was dehybridized from the triangular arch layers as described above.
- a single-stranded DNA strand called tauconnector was hybridized to the immobilized handle 1 and handle 2 using a buffer containing 40 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4 and 0.05% Tween 20 at 25 °C.
- the concentration of the connector strand and the antigen-modified spacers was 200 nM each. Further functionalization processes were identical to the preparation of the slingshot layer.
- Handle 1 5' T*AG TCG TAA GCT GAT ATG GCT GAT TAG TCG GAA GCA TCG AAC GCT GAT - Thiol 3'
- Immobilization connector 5 ' CTG CAT CAC GAG AGC TGG CAA ATG CTA CCT TTG TTG GAG GGT TCA CAC ATC AGC GTT CGA TGC TTC CGA CTA ATC AGC CAT ATC AGC TTA CGA CTA
- Biosensing measurements were performed with the DRX 2400 switchSENSE analyser instrument from Dynamic Biosensors GmbH. Fluorescent light from the Cy3-labeled DNA nucleic acid structures was excited by a green LED (517 nm bandpass filtered) that was coupled into a 5 Ox objective via a dichroic mirror.
- Emitted Cy3 fluorescence light (570 nm) was collected by the same objective, passed through the dichroic mirror and a long pass filter, and detected with a photomultiplier (Hamamatsu).
- AC square-wave signals from a frequency generator were used to actuate the DNA molecules and simultaneously trigger an event timer of a single photon counting unit (NanoHarp, PicoQuant, Germany) to record the photon arrival time with 32 ns resolution.
- a Peltier element integrated in the sample holder enabled temperature controlled experiments.
- the voltage amplitude was between 0.6 V and 1 V
- the DC offset (-0.0 V) was adjusted (app. ⁇ 0.1 V) according to the "potential of conformation transition", i.e., the inflection point in the measured voltage response curve.
- the frequency of the applied square wave potential was 10 kHz, which ensured that the DNA had enough time to stand up and lie down completely before reversing the voltage.
- the Dynamic Response is calculated from the normalized fluorescence signal F noim as
- the FRET efficiency calculated from the measured spectra is 0.7, which corresponds to the maximal efficiency anticipated under these experimental conditions (J. Phys. Chem. 122 061103 (2005), Iqbal et al. (2008) Proc. Natl. Acad. Sci. USA 105(32): 11176-11181). This suggests a hybridization efficiency close to 100%, meaning that practically all slings are hybridized to the c-spacers.
- thermodynamic properties of the DNA nanostructure melting experiments in solution were performed.
- the denaturation from a double- to a single- stranded conformation was monitored by absorption measurements at 260 nm, using the hyperchromic effect.
- Tm,l can be assigned to the melting of the 30 bp handle with an open sling, while the broader transition around Tm,2 originates from the melting of the handle in combination with a partly folded sling.
- the slingshot structure was characterized on a biosensor surface, employing the recently introduced switchSENSE method, which uses the electrical actuation of DNA nanolevers to detect proteins (Langer et al. (2013) Nature Commun. 4: 2099; Knezevic et al. (2012) J. Am. Chem. Soc. 134(37): 15225-15228) and nucleic acids in real-time (Rant et al. (2007) Proc. Natl. Acad. Sci. USA 104(44): 17364-17369). To test whether the structure is suitable for this measurement modality, the nucleic acid structures were immobilized on gold surfaces using thiol modified handles.
- Figure 3C shows that the orientation of the nucleic acid structures can be modulated efficiently by applying ⁇ 0.4V to the supporting microelectrodes (vs. an ITO counter electrode).
- the orientation switching is monitored by measuring the fluorescence emitted by Cy3 dyes attached to the center of the single-stranded loop. Whenever the negatively charged nucleic acid structures are attracted by positive potentials and lie on the surface, the fluorescence is quenched by non-radiative energy transfer to the gold film (Kaiser and Rant (2010) J. Am. Chem. Soc. 132(23): 7935-45. Conversely, when the nucleic acid structures are repelled by negative potentials and stand upright, the fluorescence emission is high.
- the hybridization of the c-spacer to surface-immobilized nucleic acid structures can be followed in real-time when incubating structures with open slings with solute c- spacers, see Figure 3D.
- the fluorescence modulation amplitude AF decreases because the sling is pulled towards the handle, which reduces the vertical extension of the standing - - structure.
- the spacer bar can be regenerated effortlessly by reloading the sling with fresh antigen-modified c-spacer. Rinsing the surfaces with NaOH solution (pH13) for app. 10 seconds suffices to denature the double strand and remove bound c-spacer. After the pH 13 -treatment the handle refolds and the sling can be hybridized with new c- spacer, as can be seen in Figure 3F which depicts 16 regeneration cycles showing little signal degradation.
- biotin bio
- digoxigenin dig
- bio-spacer-bio dig-spacer-bio
- dig-spacer-bio dig-spacer-bio
- anti-bio Fab anti-dig Fab
- anti-bio IgG anti-bio IgG
- the binding states were analyzed with a time-resolved fluorescence (TRF) measurement that allows to gauge the size of bound analyte from the switching dynamics of the nucleic acid structure on the micro-second scale (Langer et al.
- TRF time-resolved fluorescence
- Figure 4A shows TRF upward switching traces of nucleic acid structures before and after binding analytes of different sizes
- the time- resolved curves are converted to single numbers representing the switching velocity: the 'Dynamic Response' (DR) parameter equates to the integral under the curves (cf. Fig. 4A) (Langer et al. (2013) Nature Commun. 4: 2099); it decreases when analytes slow down the switching dynamics of a nucleic acid structure layer.
- DR 'Dynamic Response'
- Figure 4B exemplifies the sequential binding of anti-dig Fabs and anti-bio IgGs to a layer of nucleic acid structures loaded with bispecific dig/bio c-spacers.
- KD dissociation constant
- CF a b 10nM
- ADRFab ⁇ DRFab+Fab ⁇ DRFab+igG The bivalent IgG is an exception (cf. Fig. 4G): although its molecular weight exceeds that of two Fabs, ADRI 9 G is smaller than DRFab+Fab. This indicates that the hydrodynamic friction of a flexible construct where two Fabs are individually bound (Fig. 4E,F) to the sling is greater than that of a rigid structure where one IgG is connected to the nucleic acid structure by both arms (Fig. 4G). Overall, binding to the nucleic acid structure is found to be site- specific and does not depend on the succession of binding events.
- bivalent antibodies were investigated in order to determine if the combination product is a suitable means to realize well-defined, i.e. mono-phasic, unbinding kinetics.
- layers of bivalent probes of the present invention were compared to the conventional situation where monovalent antigen probes are randomly located on the surface.
- the two processes are dissociations of IgGs which are bound to one antigen (fast k 0 ff,i) or two antigens (slow fc 0 , 2 ), respectively.
- the bivalent IgG must interlink two antigen modified probes within its reach. This could be confirmed in previous experiments where the relative amplitudes of the slow and fast dissociations were studied as a function of the density of antigen probes. The contribution from the slowly dissociating species was found to scale with the antigen density, verifying that the slow fc 0 , 2 stems from IgGs which are doubly bound by interlinking.
- dissociation from bivalent combination products modified with two biotins proceeds in a simple, monophasic manner.
- the assembly of the triangular arch was characterized on a biosensor surface with the switchSENSE technique.
- the experiment was performed in buffer solution (40 mM NaCl, 10 mM PaPi, 0.05% Tween 20) at 25°C.
- the concentrations of solute connector strand and antigen modified spacer bar DNA was 200 nM, each.
- the hybridization of the connector strand to surface-immobilized handle 1/2 can be followed in real-time when incubating the single stranded handles with solute connectors, see figure 7a.
- the fluorescence modulation amplitude AF increases as the efficiency of the orientation modulation by the applied electric field is higher for double stranded DNA compared to single stranded DNA (Rant et al. (2007) Proc. Natl. Acad. Sci. USA 104(44): 17364-17369).
- the distance of the dyes at the top end of the handles to the gold surface increases at negative potentials during
- switchSENSE platform the association of an antibody to a antigen modified spacer bar was analyzed.
- the small molecule biotin (bio) was used as antigen and coupled to spacer 1.
- Figure 8 exemplifies the binding of an anti-bio IgG to the biotin modified triangular arch structure.
- the experiment is performed in buffer solution (40 mM NaCl, 10 mM PaPi, 0.05% Tween 20) at 25°C.
- the antibody increases the overal hydrodynamic drag of the structure, slowing the switching motion.
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